We have during the past 25 years utilized the equine infectious anemia virus (EIAV) system as an animal model for examining fundamental aspects of lentivirus replication and mechanisms of persistence and pathogenesis associated with macrophage infections. During the past grant period, we predominantly focused our efforts on developing state-of-the-art cell biology techniques to examine fundamental aspects of EIAV infection and budding, including the cloning of a functional receptor for EIAV, the determination of EIAV entry as a pH-dependent process, and the characterization of dynamic EIAV Gag interactions with actin and early and late endocytic proteins in trafficking and assembly. Based on these observations, we hypothesize that both EIAV infection and assembly/budding is mediated by similar highly concerted interactions with the dynamic cytoskeletal network and endocytic machinery via precise interactions with specific viral core proteins. We hypothesize further that HIV-1 and other retroviruses utilize similar entry and exit pathways, but that these pathways may be accessed via different portals defined by the specificity of the individual retroviral core proteins. In this competitive renewal application, we propose to test this hypothesis by addressing three specific aims: (i) to identify additional functional receptors for primary and cell-adapted EIAV strains and to define viral envelope-receptor binding determinants of receptor specificity and functional binding, (ii) to characterize the mechanisms of EIAV entry and trafficking during infection of target cells, and (iii) to define the cellular proteins and pathways adapted by EIAV for virion assembly and budding. Towards these goals we will apply our uniquely comprehensive inventory of ElAV-specific reagents and the latest imaging, molecular biology, and biochemical techniques to characterize these critical aspects of the cell biology of EIAV infection. It is anticipated that the results of these studies will provide new important insights into highly specific and crucial ElAV-cell interactions that will be applicable to other retroviruses, including HIV-1, and that will identify novel targets for the development of antiretroviral treatments. Lay Description: Theses studies are designed to define the mechanisms by which lentiviruses adapt basic cell machinery to enter and exit from target cells. This information will identify new virus-cell interactions that can be targeted for the development of novel antiviral drugs for HIV-1 and possibly other viral infections.